Our long-term goal is to understand the pathogenesis of bacterial otitis media and to develop a multivalent vaccine that reduces the incidence of otitis media caused by all major bacterial pathogens in children. [unreadable] [unreadable] Lipooligosaccharide (LOS), a major bacterial component of the outer membrane of Moraxella catarrhalis or nontypeable Haemophilus influenzae, is a possible virulence factor in the pathogenesis of otitis meda caused by the organisms. To elucidate the role of M. catarrhalis LOS in otitis media, we investigated the effects of LOS on adhesion antigens of human monocytes. M. catarrhalis LOS selectively enhanced intercellular adhesion molecule 1 (ICAM-1, or CD54) expression on human monocytes, which required surface CD14, TLR4, NF-&#954;B p65 and c-Jun N-terminal kinase (JNK) activity. Our study also revealed that the LOS induced surface ICAM-1 expression was partially mediated through a TNF-&#945; dependent autocrine mechanism and could be further augmented by lipopolysaccharide binding protein (LBP) in serum. In addition, our results also indicated that enhanced surface ICAM-1 expression on monocytes might hinder their adherence to the lung epithelial monolayer. Furthermore, the LOS activated human monocytes could stimulate adjacent nave monocytes to produce TNF-&#945; that was partially mediated via membrane ICAM-1 and IL-8/IL-8RA. These results suggest that M. catarrhalis LOS could induce excessive middle ear inflammation through a cellular cross talk mechanism during otitis media.[unreadable] [unreadable] In addition, we identified two late acyltransferase genes lpxX and lpxL responsible for lipid A biosynthesis in M. catarrhalis since Lipid A is the key biological component of the LOS molecule. Subsequently, both knockout mutants of each gene in M. catarrhalis strain O35E were constructed and named as O35ElpxX and O35ElpxL. Structural analysis of lipid A from the parental strain and derived mutants showed that O35ElpxX lacked two decanoic acids (C10:0) while O35ElpxL lacked one dodecanoic (lauric) acid (C12:0), suggesting that lpxX gene encoded decanoyl transferase and lpxL gene encoded dodecanoyl transferase. Phenotypic analysis revealed that both mutants were similar to the parental strain in their toxicity in vitro. However, the O35ElpxX was sensitive to bactericidal activity of normal human serum and hydrophobic reagents. It had a reduced growth rate in broth and an accelerated bacterial clearance at 3 h (p <0.01) or 6 h (p <0.05) after an aerosol challenge in a murine model of bacterial pulmonary clearance. These results indicate that these two genes, particularly lpxX, encoding late acyltransferases responsible for incorporation of the acyloxyacyl linked secondary acyl chains into the lipid A are important for biological activities of M. catarrhalis.[unreadable] [unreadable] Previously, we demonstrated that LOS-based conjugate vaccines elicited long lasting immunity with bactericidal antibodies in animal models. However, epitopes on the LOS recognized by the functional anti-LOS antibodies remain unidentified. To further elucidate the functional epitopes, a mutant strain D4 that was not recognized by a bactericidal anti-LOS rabbit serum in Western blotting, was generated from a serotype C strain 26404 by random transposon mutagenesis. Sequence analysis revealed there was an insertion of kanamycin resistant gene in lgt2 gene of D4 that encodes &#946;(1-4) galactosyltransferase. An isogenic lgt2 mutant 26404lgt2 was constructed. Structural analysis indicated that the mutant strain produced a truncated LOS lacking terminal galactoses from &#945;(1&#8594;6) and (1&#8594;4)-linked oligosaccharide chains of strain 26404. Further studies showed that the wild type strain specific antiserum lost the recognition to both mutant cells and their LOSs in Western blotting, ELISA, or flow cytometry assay. The antiserum also failed killing the mutant strain in a bactericidal assay, although it showed a bactericidal titer of 1:80 to strain 26404. In an ELISA inhibition assay, D-(+)-Galactose or 26404lgt2 LOS showed no significant inhibitions. However, similar to the wild type LOS, a serotype A O35E LOS with terminal galactoses only on its &#945;(1&#8594;6)-linked oligosaccharide chain showed >90% inhibition while a serotype B 26397 LOS showed >60% inhibition. These studies suggest that the terminal &#945;-Gal-(1&#8594;4)-&#946;-Gal on the &#945;(1&#8594;6)-linked oligosaccharide chain of strain 26404 LOS play a critical role in forming the epitope recognized by the functional antiserum from the conjugate vaccination.